Dendritic cells (DC) are the most powerful antigen presenting cells (APC) and play a pivotal role in initiating the immune response. In light of their unique properties, DC have been proposed as a tool to enhance immunity against infectious agents and in anticancer vaccine strategies. In the last few years, the development of DC has been extensively investigated.
Among professional antigen presenting cells (APC), DC are specialized in picking up and processing antigens into peptide fragments that bind to major histocompatibility complex (MHC) molecules. Located in most tissues, DC migrate from the periphery to secondary lymphoid organs such as the spleen and the lymph nodes, where antigen specific T lymphocytes recognize, through the T cell receptor, the peptide-MHC complexes presented by DC. While other professional and non-professional APC can only stimulate activated or memory T cells, DC have the unique capacity to prime naive and quiescent T lymphocytes.
Given their pivotal role in controlling immunity, the therapeutic role of DC has been proposed for many diseases that involve T-cell activation, such as autoimmune diseases and neoplastic disorders. Ex vivo pulsing with tumour antigens and the subsequent reinfusion of DC can lead to protection against tumours in animals. To address the efficacy of DC-based tumour immunotherapy strategies in humans, several clinical trials involving DC are currently in progress.
DC develop from hematopoietic precursor cells in the bone marrow, going through sequentially different stages of differentiation such as intermediary precursor cells in blood and immature DC in peripheral tissues and organs (Banchereau et al. 2000, Ann. Rev. Immunol. 18, 767-811). Once having reached the tissue, immature DC assume an important sensor function which is characterized by a high active uptake of antigens from the surrounding medium. Following stimulation by external signals (“danger signals”) such as bacterial or viral infections or inflammatory processes, the DC migrate into the peripheral lymphatic organs, there undergoing differentiation into mature DC, and activating T cells by presenting antigens.
DC can be obtained by differentiating progenitor cells under influence of various molecules. For example, murine bone marrow (BM)-derived progenitor cells could differentiate into myeloid DC in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). In humans, the addition of tumour necrosis factor-α (TNF-α) to GM-CSF and IL-4 was shown to induce the development of DC from bone marrow, cord blood (CB) and peripheral blood (PB) purified CD34 positive cells (CD34+ cells).
Jacobs et al (Horm Metab Res. 2008 February; 40(2):99-107) has given an overview of dendritic cell subtypes and in vitro generation of dendritic cells. The article describes the identification of different DC subpopulations including phenotypical and functional differences and describes recent developments on protocols for generation of DC. It describes that various cytokines and transcription factors are known to be responsible for the development of DC subpopulations. Depending on the subpopulation and the maturation state of these cells, they are either able to induce a broad cytotoxic immune response, and therefore represent a promising tool for anticancer vaccination therapies in humans or induce immune tolerance and are important within the context of autoimmunity.
Cytokines are small secreted proteins which mediate and regulate immunity, inflammation, and hematopoiesis. They are produced de novo in response to an immune stimulus. They generally (although not always) act over short distances and short time spans and at very low concentration. They act by binding to specific membrane receptors, which then signal the cell via second messengers, often tyrosine kinases, to alter its behavior (gene expression). Responses to cytokines include increasing or decreasing expression of membrane proteins (including cytokine receptors), proliferation, and secretion of effector molecules.
Cytokine is a general name; other names include lymphokine (cytokines made by lymphocytes), monokine (cytokines made by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action).
It is common for different cell types to secrete the same cytokine or for a single cytokine to act on several different cell types (pleiotropy) Cytokines are redundant in their activity, meaning similar functions can be stimulated by different cytokines. Cytokines are often produced in a cascade, as one cytokine stimulates its target cells to make additional cytokines. Cytokines can also act synergistically (two or more cytokines acting together) or antagonistically (cytokines causing opposing activities). Their short half life, low plasma concentrations, pleiotropy, and redundancy all complicated the isolation and characterization of cytokines.
Cytokines are made by many cell populations, but the predominant producers are helper T cells (Th) and macrophages. The largest group of cytokines stimulates immune cell proliferation and differentiation. This group includes Interleukin 1 (IL-1), which activates T cells; IL-2, which stimulates proliferation of antigen-activated T and B cells; IL-4, IL-5, and IL-6, which stimulate proliferation and differentiation of B cells; Interferon gamma (IFNg), which activates macrophages; and IL-3, IL-7 and Granulocyte Monocyte Colony-Stimulating Factor (GM-CSF), which stimulate hematopoiesis.
In addition to GM-CSF and TNF-α, a broad spectrum of cytokines has been shown to influence DC progenitor growth and differentiation. Early acting growth factors, such as stem cell factor (SCF) and Flt-3 ligand (Flt-3L) sustain and expand the number of DC progenitors whereas IL-3 in combination with GM-CSF has been shown to enhance DC differentiation. Moreover, transforming growth factor (TGF)-beta1 potentiates in vitro development of Langerhans-type DC.
In certain human dendritic lines like for instance the cell line MUTZ3, cells differentiate to DC under influence of cytokines like GM-CSF, IL-4 and TNF-alpha, whereas GM-CSF, TGF-beta1 and TNF-alpha also potentiates in vitro development of Langerhans-type DC.
Soluble factors, such as vascular endothelial growth factor (VEGF) and IL-6, inhibit the differentiation of CD34 positive progenitors into DC and redirect their development towards monocyte macrophage lineage. It is noteworthy that all these inhibitory soluble factors are secreted by cancer cells suggesting that prevention of DC development from CD34+ cells may be a mechanism of tumour escape from the immune response.
Although knowledge is accumulating with respect to how different progenitors differentiate under influence of different compounds, like cytokines to various types of DC, typically however, culturing time is long. For example, CD34 positive cells generally give rise to acceptable numbers of DC after for example 14 days of liquid culture in presence of GM-CSF plus TNF-α.
As discussed above, DC may be applied in the treatment of various diseases, including tumour diseases, infectious diseases, and autoimmune diseases. However, when DC recovered from primary cells are to be used in such treatment, the efficacy of the treatment can be severely hampered as DC or their precursor cells can only be obtained from patients or donors in very low quantities. Moreover, with the current methods, recovery requires much time and use of expensive reagents whereas the yield of obtained DC may be very small.
DC have been obtained from precursor cells, such as CD34 positive stem cells or monocytes, maturing in vitro by suitable stimulation with stimulatory molecules to form DC, although such precursor cells are extremely rare both in blood and tissue.
There is great interest in active specific immunotherapy with DC-based therapeutic vaccines for cancer. DC are intensively investigated as cellular adjuvants to harness the immune system to fight off cancer (see for example Bull Cancer. 2008 Mar. 1; 95(3):320-6.)
EP1419240 discloses the use of a cell line, MUTZ3, that can be differentiated into dendritic cells and that can be used as immunotherapeutic agent or as part of immunotherapeutic agents in the treatment of immune diseases. Although such cell line might solve the problem of the availability of sufficient cells, experimental data shows that it still requires at least 6 to 7 days of culturing under the correct conditions to obtain immature DC and at least 2 additional days for obtaining mature DC, requiring expensive cytokines to be used.
WO2006/012359 discloses a method for inducing differentiation of blood monocytes into functional antigen presenting dendritic cells. In short the cells are treated by physical perturbation, optional in the presence of for example disease effector agents.
EP1389234 discloses another method for differentiating lymphoid dendritic cells from human hematopoietic stem cells. The cells are differentiated in two steps, in a first medium comprising GM-CSF and in a second medium containing IFN-gamma. Culturing may take up to two weeks.
WO2008/036421 describes the use of an extract of reishi to increase the expression of (immature) dendritic cell markers like CD1a and CD83 in a human subject.
WO2008/02882 describes a method for producing Langerhans cells or interstitial dendritic cells from CD14 positive monocytes comprising placing said monocytes in the presence of a cell environment comprising epithelial cells and the like.
WO2004/076651 describes a method for differentiating monocytic dendritic cell precursors into immature dendritic cells comprising contacting non-activated dendritic cell precursors with a medium supplemented with GM-CSF in the absence of additional cytokines.
WO2004/083392 discloses a method for inducing differentiation of monocytes in blood into functional dendritic antigen presenting cells, using forces resulting from flow of monocytes through an apparatus having plastic channels.
US2004/0009596 describes the use of an extract from an Indian green mussel for differentiation and maturation of dendritic cells, and the extract is suggested as a replacement of GM-CSF.
US2005/0008623 describes the use of CD14 positive monocytes for obtaining various dendritic cell types. Differentiation is effected by GM-CSF and TGFbeta 1 (and IL13).
From the above it will be clear to the person skilled in the art there is need for further improvement of the available methods for the production of DC from progenitor cells. In particular there appears to be a need for methods allowing accelerated differentiation (and maturation) of DC, thereby shortening the time required for culturing and consequently reducing costs of such culturing by reducing the amount of media and for example cytokines required to obtain such functional DC.